FoxO transcription factors are phylogenetically conserved targets of insulin signaling that regulate metabolism in a variety of organisms. Reduction of FoxO1 activity prevents insulin resistance in mouse models of Type 2 diabetes, underscoring the potential utility of treating Type 2 diabetes by inhibiting FoxO1. A comprehensive understanding of how FoxO1 is regulated will facilitate the development of such therapies. Insulin inhibits FoxO by promoting phosphoinositide 3-kinase (PI3K0/Akt-dependent FoxO phosphorylation and subsequent cytoplasmic sequestration. Although FoxO regulation via phosphorylation and changes in subcellular localization is well established, evidence in multiple organisms indicates that FoxO is also regulated by PI3K/Akt-independent pathways. The molecular components and the mechanistic underpinnings of these pathways are not fully understood. By exploiting the structural and functional conservation of the insulin/PI3K/Akt/FoxO pathway throughout metazoan phylogeny and the experimental manipulability of the nematode C. elegans, we have discovered a widely expressed novel conserved protein, EAK-7, that acts in parallel to Akt to activate the C. elegans FoxO ortholog DAF-16. Human EAK-7 activates FoxO in cultured cells, suggesting that EAK-7 regulation of FoxO is also phylogenetically conserved. We hypothesize that EAK-7 is a conserved protein that activates FoxO cell-autonomously. In Aim 1, we will identify the site of action of EAK-7 in C. elegans. In Aim 2, we will assess the role of EAK-7 in regulating insulin responses in primary mouse hepatocytes. In Aim 3, we will determine the role of EAK-7 in regulating glucose and lipid metabolism in vivo. These experiments will define the role of EAK-7 in regulating hepatic FoxO1 activity, insulin responses, and metabolism in mice, potentially implicating EAK-7 as a target for pharmacologic inhibition in the treatment of Type 2 diabetes.